Topical application and formulation of erythropoietin for skin wound healing

ABSTRACT

The invention relates to the use of erythropoietin (EPO), in particular EPO in a polymer-based pharmaceutical preparation which stabilises the active compound, for the treatment of traumatised skin, in particular for wound healing in the case of mechanical or pathological injuries or in the case of burns. 
     In particular, the invention also relates to specific viscous or gelatinous formulations, in particular based on polysaccharides, preferably cellulose derivatives, which comprise EPO and are capable of stabilising the latter and releasing it slowly and uniformly to the wound.

TECHNICAL AREA OF THE INVENTION

The invention relates to the use of erythropoietin (EPO), in particularEPO in a pharmaceutical preparation which stabilises the activecompound, for the topical treatment of traumatised skin, in particularfor wound healing in the case of mechanical or pathological injuries orin the case of burns. In particular, the invention is directed to theuse of EPO in a stabilised form according to the invention based onhydrophilic polymers for stimulation of differentiation and growth ofcertain cell types, for example tissue-specific precursor cells whichare not vascular precursor cells, or of adult tissue-specific cellswhich are not endothelial cells, which are in each case encountered inthe immediate wound region in the case of skin injuries of this type.

In particular, the invention also relates to specific viscous orgelatinous formulations based on polysaccharides, in particularcellulose derivatives, which comprise EPO and are capable of stabilisingthe latter and releasing it slowly and uniformly to the wound.

TECHNICAL BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a glycoprotein hormone which controls theformation of erythrocytes from precursor cells in the bone marrow(erythropoiesis). EPO binds here to its receptor (EPO-R), which isexpressed in all haemopoietic cells.

In adults, erythropoietin is formed principally in the kidney, moreprecisely in the endothelial cells of the peritubular capillaries.Relatively small amounts are also synthesised in the liver cells(hepatocytes).

The principal action of EPO thus consists in increasing the number ofred blood corpuscles in the blood, which results in increased oxygenuptake.

In recent years, diverse authors have reported that EPO also exerts anon-haemopoietic action and EPO-R is correspondingly also expressed bycertain non-haemopoietic cells. Thus, stimulation by EPO of nerve cells,neuronal cells of the brain and endothelial cells is reported, in somecases together with direct expression of the haemopoietic EPO receptor.In other cases, the presence of a further, non-haemopoietic receptor isprognosticated, but this has not yet been proven.

Increasing importance is being accorded, in particular, to thenon-haemopoietic action, which has not been known for very long, oferythropoietin (EPO) in connection, for example, with the stimulatedformation and regeneration of endothelial and tissue cells, such asconnective tissue, muscle tissue, epithelial tissue and nerve tissue.

Thus, WO 2004/001023 describes, inter alia, the use of EPO and TPO forstimulating neovascularisation and tissue regeneration and improvingwound healing, for example after operations or injuries.

WO 2005/063965 teaches the use of EPO for the targeted, structurallycontrolled regeneration of traumatised tissue, in which not only isendothelial cell growth stimulated, but parenchymal regeneration and theformation of wall structures are also promoted, meaning that coordinatedthree-dimensional growth occurs for the construction of a functioningtissue, organ or parts thereof.

Haroon et al. (American J. Pathol. 2003, 163, 993) discuss the new roleof EPO in the context of wound healing processes induced by fibrin.

In a review article, Brines and Cerami (Kidney International, 2006)discuss the role of EPO in the protection of tissue.

Erythropoietin, and EPO derivatives or also EPO mimetics, thus appear tobe highly suitable on systemic use for specifically initiating andcontrolling neoformation and regeneration of the affected tissue in thecase of injuries to the skin, the mucous membrane, in the case of openskin and flesh wounds or also in the case of skin irritation caused byburns or scalds, and is ultimately able to promote and acceleratehealing.

WO 2005/070450 and further papers by the inventors in question describethe use of EPO in the regeneration of vessels and tissue with a weeklydose of less than 90 IU/kg of body weight (=BW), including for the areaof wound care. Although possible topical application is theoreticallymentioned here, it has nevertheless been found that systemic applicationis preferred.

It is therefore postulated to administer EPO in the case of systemicapplication in a sub-polycythemic weekly dose of less than 90 IU(international units)/kg of body weight (BW) instead of 150-300 IU/kg ofBW, as was hitherto usual for the known EPO applications. The aim isthus to achieve less stimulation of blood formation in the bone marrowregion, but, according to more recent teaching, as outlined, to enableactivation of endothelial cell progenitors in the blood region.Activation of the endothelial cell precursor cells in the blood, butalso in the tissue, and the development of endothelial cells, which formthe innermost cell layer of the blood vessels, have been connected withan improvement in vascularisation, and it is assumed that tissueregeneration is also facilitated thereby. In the meantime, this has beenconfirmed in clinical trials in the case of burn wounds.

Although some of the said papers mention topical application of EPO forthe regeneration of tissue, systemic application of the active compoundis, however, placed clearly in the foreground since, on the basis of theresults, the non-haemopoietic EPO effect found can be attributed, in theopinion of more recent papers, primarily to the newly discoveredstimulation of corresponding endothelial, vascular or CD31-positiveprecursor cells, which circulate principally with the blood stream, andonly secondarily to the growth of parenchymal tissue structuresstimulated thereby.

However, the use of EPO in a systemic application for tissue protectionor tissue regeneration is associated with significant risks owing to theside-effect potential in relation to the haemopoietic effects.

On topical application of EPO, according to teaching opinion to date,merely the inadequate distribution and reachability of the saidsystemically occurring cells or precursor cells by EPO means that onlyan unsatisfactory effect, or none at all, on tissue regeneration wouldbe observed.

In the case of regeneration of tissues after, for example, burn traumaor scalding or also in the case of ischaemic wounds, it is necessary toachieve rapid defect closure. This can only occur if the formation ofthe parenchymal components of the skin is also stimulated as quickly aspossible. The time-shifted stimulation of one component (CD31), in orderthen to facilitate the formation of another component (parenchyma),corresponds to the teaching of the authors of WO 2005/070450 and furtherpublications.

Although the formation of a vascular network can have an indirectsupporting action, it does not as such represent an end result, sincethe parenchyma is missing and under certain circumstances can only beformed secondarily and thus with a time delay.

The formation of vascular cells must thus be coordinated simultaneouslywith localised tissue formation. In this case, in accordance withconventional teaching, a dilemma occurs, since doses administeredsubcutaneously or intravenously target endothelial cell progenitorsowing to a systemic distribution principle, and sub-polycythemic dosesmust additionally be administered in order to limit side effects. Inthis case, the administration of EPO can thus only be combined withdifficulty with localised pharmacokinetics limited locally to the traumaregion of the skin.

In accordance with conventional teaching, conceptional stimulationpathways must therefore be selected, where either sub-polycythemic dosesmust be administered or repeated injections or also changes in thehalf-value period of the parent substance are necessary.

In each case, however, EPO will arrive at the actual site of need viathe bloodstream systemically distributed in the body and even furtherdiluted.

Furthermore, processes occur in the case of wounds which make topicalapplication of EPO more difficult: wound healing of, for example,injured skin or mucous membrane usually proceeds in three phases: theinflammation phase, the proliferation phase and therestoration/remodelling phase. In the case of a fresh wound or skininjury to be cared for, inflammatory processes occur within the first 24hours, which include, in particular, the immigration of diverseinflammation factors (such as, for example, fibronectin) and cells ofvarious types, such as, for example, monocytes, phagocytes, polymorphiccells and macrophages, and ultimately result in the formation of afibrin matrix and vascular endothelial cells. The wound secretion formedin the process comprises, inter alia, a series of proteolytic enzymes aswell as bacteria which have entered the wound and comprise substanceswhich act in this respect.

The proteolytic enzymes, some of which are highly active, are the reasonwhy protein- or peptide-containing medicaments, such as EPO, which havebeen applied to the wound and promote wound healing are often of littleor no efficacy, since the protein or polypeptide in question is, owingto its chemical and biological nature, deactivated, cleaved and degradedby said enzymes before it is able to develop adequate pharmacologicalefficacy. The problem is additionally exacerbated by infection of thewound with bacteria or the ingress of cell debris.

Not least for this reason, pharmaceutical proteins are generally appliedsystemically, which enables their half-value period to be significantlyextended and also enables them to be transported more quickly to thesites in the body at which they are to develop their therapeuticefficacy. However, the doses of the protein-containing active compoundmust be sufficiently high in this application method in order to achievethe desired therapeutic effect, which often inevitably results inundesired side reactions.

In the case of the therapeutic treatment of skin injuries, systemicapplication of an active compound moreover in principle appears lessappropriate, since the healing action of the medicament is actually onlynecessary locally. There is thus a general problem if protein-containingactive compounds are to be employed for the treatment of skin injuriesand open flesh and skin wounds.

The use of proteins or polypeptides in topical form, such as, forexample, EPO, which, even in plasma, has a half-value period of only 48hours, for the treatment of skin injuries of this type, as may occur inthe case of violent mechanical trauma and irritation and in the case ofburns and scalds, would, however, be very desirable in spite of theknown difficulties.

The object is thus to provide EPO or its bioequivalent derivatives,fragments, mimetics and the like for skin wound healing in the form of atopical application, without on the one hand, as just described,dramatic losses of action occurring due to proteolysis of EPO due toenzymatic or other processes in the wound, but on the other hand, aslikewise explained in detail above, possibly facilitating stimulation ofcells or precursor cells which are capable of wound healing in theimmediate wound region and if possible also in deeper tissue layers inthe vicinity of the wound by topically applied EPO, which ultimatelyshould result in faster wound closure and makes topical application ofEPO appear sensible for the first time and provides it with asignificant advantage over systemic administration.

A further aim of the invention is to create a form of administrationwhich does not have to take into account the risks and restrictions of asystemic, in particular sub-polycythemic dosage of EPO, and at the sametime can act specifically on the actual tissue-relevant cells without atthe same time excluding the localised adult endothelial cells.

SUMMARY OF THE INVENTION

Surprisingly, it has now been found that, on topical application,preferably in a suitable formulation which stabilises the activecompound and releases it uniformly, EPO is effective in the case ofinjuries or pathologically induced damage to the skin, where thisefficacy, as also shown, in particular, by histological studies, is notor virtually not attributable to the known prognosticated activation ofendothelial and other precursor cells which circulate in the blood andmay be transported to the wound—this would not be sufficient for thetarget effect—but instead is apparently based on EPO-induced stimulationof cells which have accumulated locally, i.e. in the immediate woundregion or in the extended vicinity of the wound, where, surprisingly,the cells in the skin tissue which contribute functionally to woundhealing are predominantly those which are parenchymal, i.e. are notvascular cells or precursor cells thereof and also are predominantly notendothelial cells or precursor cells thereof which are located in thewound tissue.

In general, it has been found that, in particular, the cells in thewound region which do not carry or express the endothelial cell surfacemarker CD31, or only do so to a slight extent, are stimulated todifferentiation and growth directly and locally by topically appliedEPO.

CD31 is usually found on endothelial cells or vascular cells, platelets,macrophages, granulocytes, T-cells, NK-cells, lymphocytes andfibroblasts. CD31-expressing cells are regarded as neoangiogenesisfactors. In tissues, the vessels are structures which run through thetissue like rivers, but in no way represent the tissue itself. Thesevascular cells and precursor cells thereof have surface markers, such asCD31, enabling these to be precisely differentiated and delimited fromother types of cell. Parenchymal components of the skin areCD31-negative cell types, such as keratinocytes, hair root cells anddermal cells, such as connective-tissue cells. CD31-negative cells, inparticular keratinocytes, hair root cells and connective-tissue cells,or cells which only express CD31 to a slight extent are preferably thusstimulated directly and locally by locally applied EPO. This furtheraction effect is novel and differs both from the known haemopoietic EPOeffect and also from the non-haemopoietic mechanism, which was recentlydiscovered and demonstrated systemically (see above).

It should be emphasized in this connection that, in particular, thesystemic application of erythropoietin and derivatives and analoguesthereof in the case of subcutaneous or intravenous administration onlycauses an inadequate or excessively short effect at the site of theinjury to the skin, especially as this systemic concept neglects theimportance of the tissue-specific stem cells, which are completelyindependent of vascular cells.

In accordance with the invention, the localised precursor cells in skinappendages and the stem-cell crypts of the skin can thus be stimulateddirectly by topical administration of EPO for the first time. It hasfurthermore been found that CD90-positive cells and nestin-positivecells in the wound region of the skin are also stimulated via theEPO-induced stimulation of the said substantially CD31-negative cells.CD90 is a marker protein for stem cells and neuronal precursor cells,while nestin is a marker for nerve cells. CD73-positive cells in thewound region are also co-stimulated directly by EPO. Topically appliedEPO is thus capable of stimulating virtually all important cells in theimmediate wound region which are essential for skin wound healing togrowth.

In accordance with the invention, totally irrespective of thespecifications and restrictions of sub-polycythemic dosage requirementsof the prior art, direct stimulation of essential cells and precursorcells which are specific to wound healing in the skin is effected on useof topically applied EPO, preferably in a suitable formulation based onpolymers, in particular hydrophilic polymers, and derivatives andanalogues thereof having the same action or a similar action. Thisoccurs irrespective of an action on endothelial cell progenitorsthroughout the organism which unavoidably takes place in the case of aconventional systemic application form. A systemic action of this typeis generally undesired owing to the haemopoietic effects which occur(increase in the number of red blood cells, increased blood formation inthe bone marrow, increased risk of thrombosis, etc.).

Topically and locally applied EPO in accordance with the inventionfurthermore causes no or substantially no stimulation of vascular orendothelial precursor cells circulating systemically in the bloodstream.Direct stimulation of the cells close to the wound has the advantagethat the regional skin and wound environment on the one hand restrictssystemic resorption by, for example, proteases, which reduces thedegradation of EPO before entry into the systemic circulation area andnevertheless facilitates a locally high active-compound concentration.EPO amounts which would mean a concentration of 250 IU of EPO/BW or morein the case of a conventional systemic application form can readily beadministered without the corresponding undesired EPO effects mentionedoccurring. This considerably broadens the therapeutic window for thearea of tissue regeneration. In addition, co-stimulatory effects may beinitiated by the trauma cytokines, which are only present locally inhigh concentrations, due to the topical administration of EPO.

The invention thus relates to:

-   -   the use of erythropoietin or one of its derivatives or analogues        having the same biological action (EPO), preferably in the form        of a pharmaceutical formulation which stabilises the active        compound and liberates it uniformly and is preferably based on        polymers having at least partially hydrophilic properties, for        the preparation of a medicament to be applied topically and        locally for defect closure and/or for re-epithelialisation of        traumatised skin by targeted stimulation of tissue-specific        precursor cells which are not vascular precursor cells and of        adult tissue-specific cells which are not endothelial cells and        which have in each case accumulated at a specific location in        the immediate wound region of the traumatised skin,    -   a corresponding use, where the topically and locally applied EPO        causes no or substantially no stimulation of vascular or        endothelial precursor cells circulating systemically in the        bloodstream,    -   a corresponding use, where the said cells stimulated by        topically applied EPO express no or only a very small amount of        CD31 antigen,    -   a corresponding use, where the re-epithelialisation of the skin        occurs 20 to 70%, preferably 20 to 50%, in particular 30-40%,        more quickly than by EPO in an unstabilised form under otherwise        identical conditions,    -   a corresponding use, where the re-epithelialisation of the skin        occurs 20 to 50%, preferably 30 to 40%, more quickly than by a        corresponding pharmaceutical formulation without EPO under        otherwise identical conditions,    -   a corresponding use, where the topically and locally applied EPO        stimulates the differentiation and growth of keratinocytes        and/or hair root cells and/or connective-tissue cells,    -   a corresponding use, where the topically and locally applied EPO        additionally stimulates the differentiation and growth of        CD90-positive and/or nestin-positive cells in the immediate        wound region,    -   a corresponding use, where the topically and locally applied EPO        stimulates the differentiation and growth of nerve cells and/or        neuronal precursor cells,    -   a corresponding use, where corresponding precursor cells in        deeper tissue layers in the local vicinity of the wound are        stimulated by the topically and locally applied EPO.

One of the main problems in systemic administration of EPO is to achievea sufficiently high tissue-protective action in regional areas whiletaking into account the systemic side effects on the one hand and thenecessity for establishing a high active-compound concentration on theother hand.

The aim of the invention was therefore also to create an applicationmethod and a carrier material by means of which EPO and analoguesthereof can be applied topically in a particularly effective manner.

Surprisingly, it has been found that the action effect described oftopically applied EPO and derivatives and analogues thereof having thesame biological action is particularly pronounced if the active compoundis offered for topical application in a pharmaceutical preparation orformulation which stabilises it, where the formulation or preparationshould have the further property that the active compound can bereleased to the wound as uniformly as possible and relatively slowly(slow release).

Surprisingly, it has been found that gel-forming, hydrophilic,relatively high-viscosity polymers, such as cellulose derivatives,carbomers, fatty alcohols or macrogols (polyethylene glycols) ormixtures thereof, preferably gel-forming polysaccharides, in particularfrom the group of the cellulose ethers and cellulose esters, as carriersubstance or formulation base exert a stabilising action on EPO andderivatives thereof in corresponding EPO-containing preparations to beapplied topically, such as ointments, creams, pastes or gels. The activecompound here is uniformly distributed in a viscous, swollen,polymerised or gelatinous matrix, preferably polysaccharide matrix,which protects it and from which it is, in addition, released uniformlyand slowly to the wound, in which it is able to develop its actiondirectly and immediately without significant decomposition byproteolytic enzymes taking place.

Interestingly, EPO-containing gels having a relatively high viscosity ofgreater than 20,000 mPa s, preferably greater than 30,000 mPa s, inparticular between 20,000 and 100,000 mPa s, preferably between 40,000and 60,000 mPa s, exhibit more advantageous results than lower-viscositygels having viscosity values of below 20,000 mPa s, in particular below10,000 mPa s. Such viscosity values can be achieved in gels in which thegel-forming polymer, preferably the polysaccharide gel former, has aproportion of 2-4% by weight, preferably 2-3%, but not significantlyless.

Gel formulations which comprise, as gel former, at least one swellablepolysaccharide selected from the group consisting ofhydroxymethylcellulose, hydroxyethylcellulose, carboxymethylcelluloseand carboxyethylcellulose have proven particularly advantageous.Preference is given to formulations which comprise or consist ofcarboxymethylcellulose and/or hydroxyethylcellulose.

Besides gelatinous compositions based on polysaccharides, such ascelluloses or also alginates, other swellable polymers having an overallhydrophilic action and the requisite viscous properties, as are used inmany commercially available formulations, for example gels, pastes,ointments, are in principle also suitable.

It is also possible to use hydrophobic, polymeric, generallyhigh-viscosity matrices, for example based on polyacrylates orpolyurethanes, in which hydrocolloidal particles comprisingpolysaccharides, such as carboxymethyl/ethylcelluloses, gelatine orpectins, which comprise the active compound are embedded.

The EPO is stable over a long period of more than 30 days in theadvantageous gel formulations according to the invention (FIG. 1), whilethe EPO content in an aqueous EPO solution of comparable concentration(in 0.9% NaCl) has already dropped by 80% after 20 days.

By contrast, a wound which has been treated with an EPO-containingformulation according to the invention heals about 10-50%, preferably20-40%, more quickly compared with a formulation according to theinvention without EPO. Thus, for example, wound healing of between 92and 99% can be achieved by re-epithelialisation after 4 to 8 days withan EPO-containing formulation according to the invention, whereas woundhealing with the formulation according to the invention without EPO isonly a maximum of up to 85-87% under otherwise identical conditions.

The invention thus relates to:

-   -   a viscous formulation based on at least one gel-forming,        hydrophilic polymer having a viscosity of at least 20,000 mPa×s,        which comprises erythropoietin or one of its derivatives or        analogues having the same biological action (EPO), and is        capable of releasing the latter uniformly to a hydrophilic        environment,    -   a corresponding viscous formulation which has a viscosity of at        least 30,000 mPa×s, preferably 40,000 to 60,000 mPa×s,    -   a corresponding viscous formulation which preferably comprises,        as hydrophilic polymer, a cellulose derivative, in particular a        polysaccharide selected from the group of the        hydroxyalkylcelluloses and/or carboxyalkylcelluloses, and a        hydrophobic polymer matrix based on polyacrylate or        polyurethane,    -   a gelatinous or viscous formulation based on at least one        swellable polysaccharide selected from the group of the        hydroxyalkylcelluloses and/or carboxyalkylcelluloses, which        comprises erythropoietin or one of its derivatives or analogues        having the same biological action (EPO),    -   a corresponding formulation which is obtainable by mixing EPO in        lyophilised, dissolved or suspended form with the pre-swollen        polysaccharide,    -   a corresponding formulation in which the fully swollen        polysaccharide has a viscosity of 5000-100,000 mPa×s, in        particular 20,000-50,000 mPa×s,    -   a corresponding formulation in which the polysaccharide(s) is        (are) employed in a concentration of 0.4 to 4% by weight, in        particular 2-3% by weight,    -   a corresponding formulation in which the polysaccharide is a        cellulose ether and/or a cellulose ester and is selected from        one or more members of the group consisting of:        -   hydroxyethylcellulose        -   hydroxymethylcellulose        -   carboxyethylcellulose        -   carboxymethylcellulose    -   a preferred formulation comprising, as gel former, polymerisable        hydroxyethylcellulose and/or carboxymethylcellulose,    -   a corresponding formulation which comprises EPO in a        concentration of 100 to 500 IU/g of gel formulation, in        particular 150 to 300 IU/g of gel formulation, preferably 150        IU/g of gel formulation,    -   a corresponding formulation which comprises 0.5 g to 5 g of gel        formulation/cm² of wound area, preferably 1 g to 3 g of gel        formulation/cm² of wound area, in particular 1.0 g/cm² of wound        area,    -   a corresponding formulation which, depending on the amount of        gel used, has an amount of EPO which would correspond, for a        wound area of about 100 cm², to a systemic administration of        about 50-2200 IU/kg of body weight (BW, for standard weight of        70 kg), or about 100-4500 IU/BW for a wound area of about 200        cm², and about 150-6000 IU/BW for a wound area of about 300 cm²,    -   a corresponding formulation which comprises, depending on the        wound size or wound area (1 cm² to 300 cm²), an amount of EPO of        about 50 to about 450,000 IU, on average about 500 to 300,000        IU, preferably 1500 to 60,000 IU, in particular 3000 to 10,000        IU,    -   a corresponding formulation which additionally comprises at        least one further active compound, which preferably has an        antibacterial, antiviral, fungicidal or antiinflammatory action,        acts as proteinase inhibitor or is otherwise necessary for wound        healing,    -   a corresponding formulation which additionally comprises at        least one synthetic copolymer,    -   a corresponding formulation which additionally comprises one or        more adjuvants,    -   a corresponding formulation which has been introduced into or        onto a solid carrier matrix which modulates the release of        active compound and in particular releases the EPO uniformly        into the wound region of the traumatised skin,    -   a corresponding formulation in which the solid carrier matrix is        a plaster, a foil, a film, a bandage, a gauze, but in particular        a three-dimensionally structured plaster which is capable of        stabilising and protecting the formulation itself,    -   a pharmaceutical kit of parts, comprising at least two separate        pack units, where the first unit comprises at least one        swellable polymer and the second unit comprises lyophilised EPO        or a corresponding derivative thereof.

The formulations according to the invention can be used for the topicaland local treatment of injured skin and skin diseases of various causein humans and animals. The formulations according to the invention can,in particular, also be used in dental medicine and/or for improvingwound healing in general medicine or veterinary medicine. Theformulations according to the invention can also be used in topical andlocal treatments of post-operative or post-traumatic wound infectionsand burns, in post-operative sepsis, in inflamed and/or infected ulcers,in chronic ischaemic wounds, in acute and chronic skin infections ordermatoses, in acne, rosacea, psoriasis or in mucous membraneulcerations, or mucous membrane injuries, such as mucous membrane bonewounds, in particular in the jaw or dental area.

The invention thus also relates to the use of the formulation specifiedabove and below for the preparation of a medicament for the topical andlocal treatment and healing of wounds by re-epithelialisation of theskin, in particular for the treatment and healing of burn wounds or ofskin, mucous membrane and bone wounds and wounds in the dental area.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Stability Study of EPO-Containing Preparations

As can be seen in FIG. 1, the EPO content varies in the range 90-100% ofthe nominal content (150 U/ml) during the investigation, meaning thatthe preparation and also the corresponding hydrogel can be assumed to bestable over a period of 4 weeks after preparation.

By contrast, a significant degradation of EPO was apparent in the sodiumchloride-containing solution (0.9% by weight of NaCl in water comprising40 U/ml of EPO) over the experiment period, meaning that only between 20and 30% of the nominal content were found after three and four weeks.

FIG. 2:

The figure shows the re-epithelialisation of the skin, in %, oftraumatised rats which had been treated with a formulation according tothe invention (3% of carboxymethylcellulose) without EPO(control/placebo, gel F—left-hand bar) and with EPO (about 0.3 g of gel:150 IU/g of gel=about 200 IU/BW, gel E—right-hand bar) for 4 days.

The results are shown in tabular form (top picture) and graphically(bottom picture).

FIG. 3:

The figure shows the re-epithelialisation of the skin, in %, oftraumatised rats which had been treated with a formulation according tothe invention (3% of carboxymethylcellulose) without EPO(control/placebo, gel F—left-hand bar) and with EPO (about 0.3 g of gel:150 IU/g of gel=about 200 IU/BW, gel E—right-hand bar) for 8 days. Thebandage/plaster was changed after four days, and the wound was treatedwith fresh gel. After treatment with the formulation according to theinvention for 8 days, the skin is virtually 100% restored.

The results are depicted in tabular form (top picture) and graphically(bottom picture).

FIG. 4:

FIG. 4 depicts the histological investigation of wound healing aftertreatment with a gel as described in Example 8 for 8 days. Cells whichexpress CD31 protein and nestin have been visualised by appropriatestaining.

The results show that the layer thickness of the epithelium issignificantly increased on treatment with EPO compared with the controlformulation. In addition, it can be seen that neoformation of theepithelium is essentially not correlated with an increase inCD31-positive (stained) cells, but is to a certain extent withnestin-positive cells.

FIG. 5:

FIG. 5 is the photographic evidence of the healing of a third degreechronic ischaemic ulcer of the lateral malleolus in a diabetic patientafter local treatment three times with the formulation according to theinvention comprising 3000 IU of EPO (in total 9000 IU). The bottompicture shows the course after 15 days (Example 16).

FIG. 6:

FIG. 6 shows a photograph of the healing process of a thermal woundafter removal of a split-skin graft in a patient by treatment with asingle administration of 3000 IU of EPO in a hydrogel according to theinvention (Example 17).

DETAILS OF THE INVENTION

It has been found that rapid defect closure can be achieved in the caseof skin regeneration, for example after burn trauma, scalding, ordisease-induced wounds by topically applied EPO, in particular in theform of a gelatinous formulation described in greater detail above andbelow, in particular a hydrogel based on cellulose. Furthermore,re-epithelialisation of the wound region is crucially promoted bytopically administered EPO. This is evident from a greater layerthickness of the epithelium (FIG. 4). This can only occur if theformation of the parenchymal components of the skin is also stimulatedas rapidly as possible.

It can furthermore be observed that the formation of a vascular networkis also initiated by topically applied EPO, but this is associated withlocal formation of the parenchyma, in contrast to the known teachingthat wound healing caused by EPO is firstly set in motion by stimulationof vessel-forming cells, i.e. EPO merely causes vascular formation andthe formation of parenchymal tissue commences indirectly and at best ina time-delayed manner, as a consequence of EPO stimulation ofendothelial cells and precursor cells thereof.

However, the results of the present invention clearly show that theformation of vascular cells is coordinated at least at the same timewith localised tissue formation.

It can furthermore be observed in accordance with the invention that thetopical application of EPO causes tissue-specific precursor cells whichare nestin-positive to be found to an increased extent in the boundaryregion of the new epidermis. Neuronal stem cells are thus stimulated inthis region, which explains the improved sensitivity of the skin duringwound healing.

For the formulations according to the invention, use is preferably madeof recombinant erythropoietin, as is commercially available. However, itis also possible to employ in accordance with the invention EPOderivatives which have been developed in order to extend the half-valueperiod of the active compound in the blood or in the serum compared withnative EPO. EPO derivatives of this type can also be employed fortopical application, although this is not absolutely necessary since thegelatinous formulations according to the invention develop their ownspecific protective action against normal EPO. These include, forexample, pegylated EPO, EPO with a modified glycosylation pattern (forexample Aranesp®), silylated EPO, or EPO which has been fused to otherpolypeptides or fragments of immunoglobulins (for example to the Fc partof an antibody) (known, for example, from WO 02/49673 or WO 01/02017).Furthermore, biologically active synthetic EPO peptide mimetics (asknown, for example, from WO 96/40749, WO 96/40772, WO 01/38342, WO01/091780, WO 2004/101611, WO 2004/100997, WO 2004/101600, WO2004/101606 and WO 2006/050959) can also be employed. Since thesemimetics usually have significantly shorter amino acid chains and thusgenerally undergo faster degradation, they experience the requisite andadequate stabilisation by the formulations according to the invention.

In the case of the known systemic use of EPO, between 150 and 300 IU/kgof body weight (BW) of the active compound are generally applied perdose. At significantly higher doses, the undesired side effects becomedominant. In the case of an adult human (70 kg), this corresponds tobetween 10,500 and 31,500 IU of EPO/dose. This in turn corresponds toabout 80 to 250 μg of EPO/dose (13,000 IU˜100 μg of EPO).

In the case of the topical use according to the invention for injuriesto the skin, significantly larger amounts of EPO can be employed perdose by means of the formulations or gels according to the inventionwithout significant side effects occurring. Thus, for wounds ofcorresponding size (for example 300 cm²), EPO amounts which wouldcorrespond to about 6500 IU/kg of body weight (BW) in the case ofsystemic administration can be employed. Such an amount of EPO appliedsystemically would no longer be tolerable. The requisite total amountsof EPO vary considerably depending on the wound size and amount of gelfor a wound area and content of EPO/g of gel.

In general, the dose can be regarded in accordance with the invention asthe amount of EPO which is required for single treatment of a wound (ofvarious size). Since a wound is defined not only by its area, but alsowhere appropriate by its depth, the dose of requisite active compoundcan vary greatly, since the wound is preferably filled with theformulation according to the invention.

If the body weight for an adult human is taken as 70 kg, the followingvalues, for example, arise for wound healing:

50-500 IU/BW→3500-35,000 IU of EPO per dose or wound and wound size;50-150 IU/BW→3500-10,500 IU of EPO per dose or wound and wound size;100-500 IU/BW→7500-35,000 IU of EPO per dose or wound and wound size;150-300 IU BW→10,500-21,000 IU of EPO per dose or wound and wound size.The wound sizes and gel concentrations that correspond to these valuesare shown in Table 1.

The low EPO units are preferably employed for small wounds having awound area of about 5-20 cm², while the upper values are used for largewounds having an area of around 100 cm² or larger. In the case of woundshaving an area of, for example, 10 cm², EPO amounts of between about 500and 15,000 IU can be employed in accordance with the invention. In thecase of wounds having an area of, for example, 100 cm², about5000-150,000 IU of EPO are required. This means that on average about50-1500 IU of EPO/cm² of wound area, preferably 75-450 IU of EPO/cm²,should be applied in accordance with the invention to the wound forre-epithelialisation and defect closure of the wound. The amountsapplied may of course be significantly above or below the said valuesunder certain conditions.

The above-mentioned values are based on an EPO content of 100-500 IU/gof gel formulation, in particular 100-300 IU/g of gel formulation,preferably 150-200 IU/g of gel formulation. In detail, the followingranges of EPO amounts are employed in accordance with the invention pergram of formulation base:

100 IU/g of gel-300 IU/g of gel, 100 IU/g of gel-150 IU/g of gel, 100IU/g of gel-200 IU/g of gel, 100 IU/g of gel-250 IU/g of gel, 150 IU/gof gel-200 IU/g of gel, 150 IU/g of gel-250 IU/g of gel, 150 IU/g ofgel-300 IU/g of gel, 200 IU/g of gel-250 IU/g of gel, 250 IU/g ofgel-300 IU/g of gel.

In general, about 0.5 to about 5 g, preferably 1-3 g, in particular 1 g,of gel according to the invention are used per square centimetre (cm²)of wound area in an adult, depending on the injury. However, the amountsused may be above or below these concentrations. In particular, thefollowing gel concentration ranges are employed: 0.5 g of gel/cm²-2.0 gof gel/cm², 0.5 g of gel/cm²-1.0 g of gel/cm², 1.0 g of gel/cm²-2.0 g ofgel/cm².

Formulations according to the invention thus comprise, for example,approximately the following amounts of EPO (IU) or the amounts of EPOindicated in Table 1 per dose, corresponding to the wound size and theamount of EPO per gram of formulation base: 2000 IU, 3000 IU, 4000 IU,5000 IU, 6000 IU, 7000 IU, 8000 IU, 9000 IU, 10,000 IU, 12,000 IU,15,000 IU, 20,000 IU, 25,000 IU, 30,000 IU, 35,000 IU, 40,000 IU, 45,000IU, 55,000 IU, 60,000 IU, 70,000 IU, 80,000 IU, 90,000 IU, 100,000 IU,120,000 IU, 135,000 IU, 150,000 IU and 180,000 IU.

TABLE 1 IU of EPO IU of EPO IU of EPO IU of EPO IU of EPO cm² of Amountof (100 IU/g of (150 IU/g of (200 IU/g of (300 IU/g of (500 IU/g ofwound area gel (g) gel) gel) gel) gel) gel) (A) 0.5 g of gel/cm² ofwound area 1 0.5 50 75 100 150 250 10 5 500 750 1000 1500 2500 100 505000 7500 10,000 15,000 25,000 200 100 10,000 15,000 20,000 30,00050,000 300 150 15,000 22,500 30,000 45,000 75,000 (B) 1 g of gel/cm² ofwound area 1 1 100 150 200 300 500 10 10 1000 1500 2000 3000 5000 100100 10,000 15,000 20,000 30,000 50,000 200 200 20,000 30,000 40,00060,000 100,000 300 300 30,000 45,000 60,000 90,000 150,000 (C) 2 g ofgel/cm² of wound area 1 2 200 300 400 600 1000 10 20 2000 3000 4000 600010,000 100 200 20,000 30,000 40,000 60,000 100,000 200 400 40,000 60,00080,000 120,000 200,000 300 600 60,000 90,000 120,000 180,000 300,000 (D)3 g of gel/cm² of wound area 1 3 300 450 600 900 1500 10 30 3000 45006000 9000 15,000 100 300 30,000 45,000 60,000 90,000 150,000 200 60060,000 90,000 120,000 180,000 300,000 300 900 90,000 135,000 180,000270,000 450,000

In accordance with the invention, the novel formulations comprise atleast one gel-forming, viscous, hydrophilic polymer, preferably acellulose derivative, preferably a polysaccharide, or alginates orderivatives thereof, chitin or derivatives thereof or salts thereof, orstarch. The origin of the gel-forming polysaccharides is unimportanthere, i.e. these gel-forming polysaccharides can be of vegetable oranimal origin or prepared synthetically. It is also possible to usepolysaccharides which are of vegetable or animal origin and have inaddition been modified by chemical synthesis.

Cellulose derivatives are preferably employed for the gels orformulations according to the invention. The group of the cellulosederivatives in connection with the present invention includes, inparticular, cellulose ethers and cellulose esters and salts thereof.Cellulose ethers used here are, in particular, hydroxyalkylcelluloses,such as, for example, hydroxymethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose or hydroxybutylcellulose, but in particularhydroxymethylcellulose or hydroxyethylcellulose. Cellulose esters usedhere are, in particular, carboxyalkylcellulose, in particularcarboxymethylcellulose, carboxyethylcellulose, carboxypropylcellulose orcarboxybutylcellulose, but preferably carboxymethylcellulose orcarboxyethylcellulose, where carboxymethylcellulose is the mostpreferred.

According to a further embodiment, the formulation according to theinvention may also comprise at least two different gel-formingpolysaccharides of those mentioned above, in particular of the cellulosederivatives mentioned above. It has proven particularly advantageoushere for at least one compound from the class of the cellulose ethersand at least one compound from the class of the cellulose esters to beemployed. Particular preference is given here to a formulation/hydrogelwhich comprises, as gel-forming polysaccharides, hydroxyalkylcelluloseand carboxyalkylcellulose, in particular hydroxyethylcellulose andcarboxymethylcellulose.

Furthermore, a gel according to the invention may in particularcomprise, as gel-forming polysaccharide, at least one water-solublecellulose derivative. These are distinguished by the fact that they donot form swollen particles within the gel, in turn resulting in a veryhomogeneous hydrogel. In addition, a water-solublepolysaccharide-containing gel'exhibits particularly good spreadabilityon application to a wound, in addition forms a particularly smoothsurface and can be modelled particularly well. Water-soluble cellulosederivatives which are not crosslinked are particularly suitable here.

The formulations according to the invention may furthermore comprise astructure former or a viscosity-modulating compound. Polyacrylic acidand salts thereof and in particular crosslinked polyacrylates areparticularly suitable for this purpose. Examples of suitable acrylatesof this type are: (poly)methacrylate, (poly)methyl methacrylate,polyacrylamide, (poly)ethoxyethyl methacrylate. These polyacrylic acidderivatives additionally have the advantage that they are able to takeup a considerable proportion of their own weight in water. Thecombination of these acrylic acid derivatives with at least onegel-forming polysaccharide thus enables the specific preparation of agel whose water uptake and water release capacity can be controlled. Theratio between cellulose derivative and acrylic acid derivative in theformulation can be between 20:1 and 1:1, but preferably between 10:1 and2:1.

Furthermore, the formulation according to the invention can be based ona viscous polymer which overall has a hydrophilic action and which has ahydrophobic carrier or supporting matrix, for example comprisingpolyurethane or polyacrylate, in which said polysaccharides comprisingthe active compound, such as, for example, carboxyalkylcelluloses, areembedded. Hydrogels of this type are particularly suitable for woundhealing since they keep the wound moist, create an optimum environmentfor autolytic debridement, and deposit wound secretions in the secondarybandage. An example thereof is Varihesive® hydrogel.

In general, other hydrophobic polymer backbones or supporting structuresin which hydrocolloidal particles of a polysaccharide, as indicatedabove, are embedded are also possible.

In principle, all gel and ointment bases based on polymers, inparticular built up on the basis of polysaccharides, and which haveproven particularly suitable for wound healing and are commerciallyavailable are suitable for the formulation according to the invention solong as uniform slow release of EPO by maintenance of viscosity valuesas described above and below is ensured.

The formulations according to the invention may furthermore additionallycomprise electrolytes. Suitable electrolytes in connection with thepresent invention are compounds which are capable of dissociation intoions, in particular on dissolution in water, and are built up frommono-, di- and/or trivalent ions. These electrolytes can be, forexample, in the form of inorganic or organic salts and differ from anypolymers having an ionic character which may be present in theformulation. Particularly suitable in this connection are chlorides,iodides, sulphates, hydrogensulphates, carbonates, hydrogencarbonates,phosphates, dihydrogenphosphates or hydrogenphosphates of the alkali andalkaline-earth metals, but in particular sodium chloride, potassiumchloride and calcium chloride. These electrolyte mixtures simulateparticularly well the electrolyte mixture in a wound serum released by awound. These electrolyte-containing formulations according to theinvention thus provide the wound with a sphere which promotes woundhealing to a particular extent.

In a further alternative embodiment of the invention, the formulationaccording to the invention additionally comprises a polyol. This polyolis highly suitable as moisture donor and thus represents a carecomponent for the skin surrounding the wound. Particularly suitable forthis purpose are polyols which can be selected, for example, from thegroup consisting of: glycerin (glycerol), glycol, propylene glycol,polyethylene glycol, polypropylene glycol, polyethylene-propylene glycolor mixtures thereof. In particular, the polyol employed in the presenthydrogel can be glycerin or polyethylene glycol and mixtures thereof inamounts of between 0.5% and 10% (w/w), based on the entire gel.

The formulations or gels, in particular hydrogels, according to theinvention must have a viscosity which is suitable for protecting EPO inan optimum manner, but at the same time ensure that the active compoundcan be released to the wound in sufficient amount and sufficientlyquickly without previously being metabolised.

Surprisingly, it has been found that this is ensured in an optimummanner only by means of formulations which have a relatively highviscosity, which is achieved with a proportion by weight of swellablepolymer in the gel as a whole, preferably of cellulose derivatives, ofat least 1.5%, but preferably 2-4%, very particularly preferably2.5-3.5%, in particular 3% or around 3%. Gels having such a viscosityare generally rarely employed for medical purposes, since they usuallycannot be processed as well as gels having a gel former content of 1-2%.As mentioned above, the use of, for example, acrylates enables theprocessability of the gel to be improved.

The gels or formulations according to the invention based on hydrophilicpolymers, such as, for example, cellulose ethers and/or celluloseesters, for example carboxyalkylcelluloses, thus have a dynamicviscosity of 5000 to 100,000 mPa s, preferably greater than 20,000 mPas, in particular 20,000 to 70,000 mPa s and very particularly 40,000 to60,000 mPa s (measured in Bohlin model CSR—10 rheometer, 4°/0.40 mmconical spindle, oscillometric measurement, T=22-25° C.). A hydrogel ofthis type can be distributed sufficiently well and uniformly over and ina wound, has good coherence even on uptake of wound exudate and does notrun out of a wound to be treated.

It is provided that the preferred EPO-containing formulations or gelsaccording to the invention have the following composition: at least 70%by weight of water and 1.5-6.0% by weight of gel-forming polysaccharide,preferably at least one cellulose derivative, in particularcarboxymethylcellulose and/or hydroxyethylcellulose, and optionally0.1-10% by weight of acrylic acid derivative and/or 1-20% of polyol,preferably 1-5% by weight of glycerol, and/or 0.1-5% by weight ofelectrolyte.

The formulations according to the invention may optionally compriseadjuvants known per se for the gel preparation, including suitablepreservatives.

The formulations or gels according to the invention may also compriseother active compounds which are necessary as such for wound healing.This is particularly appropriate if, for example, bacterial, viral orfungal infections have occurred or prophylaxis against such infectionsis intended. Corresponding antibiotics, antimycotics or alsoantiphlogistics which are suitable for this purpose are described in theprior art. In particular, active compounds which can be employedtopically are suitable here. Examples of antibiotics which can beemployed topically are tetracyclins or penicillins or also erythromycin,bacitracin, tyrothricin, colistin and polymyxin B or aminoglycosides,such as neomycin, kanamycin and paromycin or mupirocin.

In a particular embodiment, at least one proteinase inhibitor, which isintended to inhibit the proteinases which occur to a massive extent inthe wound secretion, is added to the formulation according to theinvention. A particularly suitable proteinase inhibitor in accordancewith the invention is aprotinin, which is capable of inhibiting thepro-inflammatory cytokine secretion which occurs in a wound.Surprisingly, it has been found that EPO is particularly effective forwound healing on topical administration in the presence of aprotinin.

Furthermore, the gels or formulations according to the invention mayalso comprise disinfectants, such as, for example, povidone-iodine orthe like.

Besides the EPO-containing formulation according to the invention assuch, the present invention also relates to a wound dressing whichcomprises a medical carrier material and the formulation or gel of thetype described. For example, the carrier material employed herecomprises nonwovens or knitted or woven textile fabrics made fromnatural or synthetic fibre materials, but also film coverings, forexample in the form of a spray plaster. The medical carrier materialhere is coated or impregnated with the gel or hydrogel on one or moresides.

A very particularly suitable wound dressing can be provided in the formof a special plaster which comprises the active compound EPO in aparticularly optimised manner and releases it to the wound.

A plaster of this type is described in detail in EP 08 011 985.2 andcomprises a carrier matrix which comprises the active compound(s), wherethe carrier matrix has the following to structure:

(i) areas or structures in the form of one or more cavities serving ascontainer for the medicament, and (ii) areas or structures in the formof one or more cavities serving as channel-like structures, which servefor the uptake and draining of wound secretions and/or aeration and/ortopical introduction of further active compounds and/or of cells whichpromote skin healing, where (a) at least one area in accordance with (i)is adjacent to at least one area in accordance with (ii), (b) areas (i)and (ii) are sealed on the side facing away from the wound, (c) areas(i) and (ii) are open or at least permeable to the active compounds,wound secretions, further active compounds/said cells on the side facingthe wound, (d) the areas (i) and (ii) in the form of cavities arearranged in the plane of the plaster surface, and (e) the cavities inareas (ii) have at least one opening or connection for a syringe orsuction/supply device which enables either the drainage of woundsecretions which have accumulated in the cavities, with generation of areduced pressure in the plaster, and/or optionally the application ofsaid further active compounds.

A corresponding EPO-containing plaster may furthermore be designed insuch a way that, in the case of weeping skin or wounds or skin or woundswhich produce wound secretions, (f) the cavities in areas (ii)additionally have drainage means which enable wound secretions whichhave accumulated in the cavities to be drained. In this case, thecavities in areas (ii) may be channels which are connected to oneanother. The cavities in areas (i) may be trough-shaped, where the openside of the troughs faces the wound, and the troughs have a rectangular,square, hexagonal/honeycomb-shaped or round base area. The said cavitiesin areas (i) are separated from one another by bridges of the materialof the carrier matrix. The bridges themselves may likewise havechannel-like structures, at least some of which are connected to oneanother and which serve for the drainage of wound secretions and/oraeration and/or the supply of further active compounds/said cells.

In addition, the cavities in areas (i) serving as depot for themedicament represent sub-structures arranged in clusters, which areseparated from one another by bridges of the material of the carriermatrix. The cluster domains have any desired rectangular, square,hexagonal/honeycomb-shaped or round base area shape, in particular asquare or rectangular base area shape, where the shape of the clusterbase area may, if desired, be identical to or different from the basearea shape of the sub-structures of areas (i). It may be provided herethat a cluster domain of area (i) is adjacent to at least one cavity ofarea (ii) and that, if desired, two cluster domains of areas (i) areseparated from one another by a cavity of area (ii).

The formulations/gels according to the invention, which are optionallyapplied to a wound dressing which is known in principle and iscommercially available, or, for example, like the plaster describedabove, can be changed every 12, 24, 48 or 72 hours, preferably every 48hours, which may principally be necessary due to other factors andcircumstances, such as, for example, the formation of wound secretions,bleeding or infections arising, which have to be treated. Merely forreasons of stability and activity of the active compound EPO within theformulation base according to the invention, however, it is notnecessary to change the bandage or plaster before complete woundhealing.

The gels according to the invention can be prepared by methods known perse. One of these methods is described in greater detail in the examples.

The EPO-containing gels according to the invention can, as indicatedabove, be employed, in particular, in the treatment of wounds, inparticular to the skin, the mucous membrane, in the dental area, in thecase of mucous membrane/jaw injuries in the oral cavity, or in the caseof burns or scalds of the skin or skin diseases accompanied by chronicwounds. Wounds to the skin may be caused, for example, by cuts,punctures, crushing, bites or shot injuries, or may arise as anunavoidable consequence of operations or tooth extractions. Furthermore,diverse diseases may cause wounds to the skin or flesh, or form openulcers. Relatively large wounds also arise in the case of organtransplants or amputations and have to be provided with therapeutic caretopically and locally.

In the dental area, relatively small wounds may also arise in the caseof carious inflammation and periodontitis and can successfully betreated with the formulations/gels according to the invention. If theenamel surface of the tooth is damaged, bacteria penetrate further intothe underlying dentine. Pulp processes are present in the radial dentinetubules, meaning that partial or total infection and thus inflammationof the pulp then occurs. If no treatment is given, the consequence isdeath of the pulp tissue (necrosis) and bacterial decay (gangrene). Ifthe gangrenous masses are not removed, inflammation outside the root tipis the consequence. Granulomas, cysts, fistula formation or abscessesmay develop. EPO or the EPO-containing gel can successfully be employedat each of these stages, advantageously after correspondingantibacterial treatment.

The formulations or EPO-containing gels according to the invention areparticularly suitable for the treatment of comparatively deep wounds andcan be employed extremely well as wound fillers. Thus, for example, deepdermal ulcers, which very frequently weep heavily, can be treated withthe gels according to the invention. The relatively high viscosity ofthe gel prevents liquid from trickling out of the wound, or at leastreduces this. In addition, however, dry wounds, such as, for example,dry ulcus cruris, can also be treated with the present gel. In thiscase, the present gel exhibits its ability to supply the wound withliquid and to ensure the removal of undesired substances, deposits andnecroses by gentle debridement. Other types of wound for which theformulation according to the invention can be used include, but are notlimited to, stage I, II, III decubitus ulcers (pressure sores), ulcuscruris (leg ulcer, leg sore), diabetic foot syndrome, skin ulcers, bloodulcers, first and second degree burns, grazes and chronic wounds.

Finally, the invention relates to a kit of parts which comprises atleast two separate packs, where the first pack comprises the materialsof the hydrogel or constituents of the hydrogel, such as polysaccharideand/or polymeric (hydrophobic) supporting matrix, and the second packcomprises the active compound EPO as stable lyophilisate. The kit ofparts may also comprise the constituents of the formulation base, ifappropriate, in different packs. A pack containing an aqueous componentmatched to the amount, in which the solid components can be dissolved orsuspended, may likewise be a further constituent of the kit of partsaccording to the invention, i.e. the pharmaceutical kit may include afurther pack unit which comprises the solvent necessary forswelling/polymerisation (preferably water or water-containing agents).The kit according to the invention may comprise the swellable polymer inthe form of a powder or in pre-swollen form, where, in the latter case,the viscosity of the pre-swollen polymer should be kept sufficiently low(preferably below 5000 mPa s) that uniform mixing with the activecompound EPO from the second pack unit is ensured. After addition ofEPO, the carrier polymer/gel can finally swell to completion with thedesired viscosity.

The individual separated packs may also be physically connected to oneanother in such a way that their contents can be mixed with one anotherby simple mechanisms, such as piercing, puncturing, forcing through,tearing off or the like, for example in the form of a mixed closuresystem. In this way, it is possible to delay addition, in particular, ofthe active compound EPO, which has a very long shelf life in lyophilisedform with retention of its activity, to the still not fully polymerisedgel or formulation base until preparation of a fresh gel in thepre-swollen state of low viscosity in accordance with the inventionimmediately before application to the wound, meaning that losses ofactivity cannot occur, as may be the case under certain circumstances inactive compound-containing formulations which are completely finishedfrom the beginning.

The following examples are intended to describe the invention in greaterdetail, but without restricting it in any way. In particular, the personskilled in the art will be able, if desired, to generalise the knowledgefrom these examples with the aid of his general knowledge.

Furthermore, the substances indicated in the examples, including theirparameters, properties, physical quantities, data, and the specificmethods described, which the person skilled in the art, unless statedotherwise or prevented by sensible or technical/scientific reasons, willalso be able to generalise and bring into other connections thanindicated in the examples, should merely be regarded as illustrative.

EXAMPLES Example 1

Erythropoietin (EPO) is used in the form of the finished medicament(NeoRecormon 10,000 TU, powder and solvent for the preparation of aninjection solution in cartridges, batch MH68260 08, PZN 742 914 3, RocheReg. Ltd., Welwyn Garden City, UK).

The active compound is converted into a pre-sterilisable preparationunder aseptic conditions using the following gel formers:

-   -   Hydroxyethylcellulose Ph. Eur. 5.1 (trade name:        Hydroxyethylcellulose 250 HX Pharm, batch 06E29-N01, Fagron,        D-Barsbüttel)    -   Carmellose-sodium/carboxymethylcellulose Ph. Eur. 5.0 (trade        name Tylopur C600, batch 516 762 65, Caelo, D-Hilden)    -   Methylcellulose/hydroxypropylmethylcellulose USP (trade name        Metolose 90 SH-100, batch 206314, Shin-Etsu, D-Mülheim)    -   Povidon Ph. Eur. 5.0 (trade name Kollidön 25, batch 74-0915,        BASF, D-Ludwigshafen)    -   Varihesive®, Convatec, Varihesive E® is a hydrocolloid bandage.        It consists internally (lying on the wound) of a hydrocolloid        layer based on a swellable polysaccharide which is embedded in        an adhesive polymer matrix, and externally of a film-coated        polyurethane foam.        Besides water for injection purposes as bulk (Ph. Eur. 5.0),        glycerol (85% or anhydrous, batch 058 006 2, Fisher Scientific,        Loughborough, UK) is used as adjuvant. The primary packaging        employed for accommodation of the formulations is Fiolax        injection vials, glass type I (Münner-städter Glaswarenfabrik        GmbH, D-Münnerstadt). These are covered with 20 mm Pharma-Fix        sealing discs and crimp-sealed with 21 mm metal crimp seals        (both VWR, D-Hannover). The release studies are carried out        using organotypical skin models, whose production, cultivation        and use were carried out in accordance with the following        reference: C. Hoffmann, C. C. Müller-Goymann, “Use of artificial        skin constructs in permeation studies of clindamycin phosphate”,        Pharmazie 60 (2005) 350-353.

Example 2 Formulation of Hydrogels Containing No Active Compound

The gel former to be investigated is weighed out into a tared mortarunder low-germ conditions and ground with the intended amount ofglycerol. After addition of water for injection purposes as bulk inportions, the preparation is allowed to swell for at least 2 hours,before evaporated water is replaced and the hydrogel is homogenised inthe mortar. The hydrogel is transferred into an injection vial which hasbeen sterilised with hot air in 3.3 g portions, which can be carriedout, depending on the consistency of the preparation, by drawing up bymeans of a syringe or by transfer into an Unguator jar and subsequentdispensing with the aid of an attached Unguator applicator (both GakoKonietzko GmbH, D-Bamberg). The sealed injection vial is then sterilisedby means of saturated, live steam under standard conditions of Ph. Eur.

Example 3 Evaluation of the Rheological Properties of the HydrogelsContaining No Active Compound

In order to be able to estimate the thermal degradation of the gelformers during autoclaving, sterilised hydrogels are assessed visuallywith unsterilised samples and measured in a plate-and-cone viscometer(GVO rheometer, Bohlin Instruments, Cirencester, GB) at a temperature of20.0° C. and a cone apex angle of 1°. The final gel former content isdefined in accordance with the therapeutic demands made of theformulation.

Example 4 Preparation of an EPO-Containing Gelatinous Formulation

In each case, 500 IU of EPO are injected into the cooled andpre-sterilised hydrogel from the reconstituted EPO 10,000 IU solutionunder a clean bench; to this end, use is made of a Reco-Pen (RocheDiagnostics GmbH, D-Mannheim), which releases the requisite medicamentsolution via an attached 12 mm Clickfine universal needle (Ypsomed,D-Sulzbach). Taking into account the EPO volume of 50 μl transferred for500 IU of EPO, an active-compound content of 150 IU/g is obtained in thepreparation. In order to be able to ensure homogeneous distribution withexclusion of the risk of protein denaturing, the EPO solution wasallowed to diffuse into the hydrogel over 24 hours. For improved mixing,the injection vial was rotated twice through 180° in this period. Thepreparations were stored at +2 to +8° C. with protection against light.In detail, the following preparation procedure is used by way of examplefor a hydrogel having an erythropoietin content of 150 IU/g of gel:

-   -   Preparation of the gel containing no active compound under        low-germ conditions    -   Grind carboxymethylcellulose 250 HX with glycerol (2.4%) in a        mortar    -   Incorporate water in portions    -   Allow to swell for at least 2 hours    -   Replace evaporated water, homogenise. Alternatively, an        electronic stirring system (e.g. Cito-Unguator 2000) can also be        employed for the preparation of the gel containing no active        compound    -   Transfer gels in portions of 3.3 g each into an injection vial        of glass type 1 which has previously been cleaned and sterilised        with hot air, and crimp-seal, e.g. using a syringe or Unguator®        applicator    -   Autoclave crimp-sealed gel under standard conditions    -   Bring injection vials to a temperature of +2 to +8° C. before        further processing    -   Transfer 500 IU of EPO into each injection vial under a clean        bench, e.g. from NeoRecormon 10,000 E two-chamber cartridges        using the Reco-Pen with release of 2 pen units via a Clickfine        universal needle    -   Allow EPO to diffuse in over at least 24 h, rotate the gel twice        through 180° in the injection vial during this period for better        mixing.        Gels are prepared analogously using hydroxyethylcellulose,        carboxyethylcellulose, carboxypropylcellulose,        hydroxypropylcellulose (between 1.5% and 3.5% (w/w) cellulose        content).

Example 5 Stability Testing of EPO-Containing Formulations

In order to test the stability of erythropoietin in preparations,samples are taken at time 0, 1, 2, 3 and 4 weeks after preparation ofthe respective preparations and sent for erythropoietin analysis.Throughout the experiment period, the storage conditions mentioned aboveare maintained. In addition, a preparation having an erythropoietincontent of 40 IU/ml in 0.9% aqueous sodium chloride solution is alsoincluded in the study. The results are shown graphically in FIG. 1.

Example 6 Active Compound Release Experiments Through Skin Constructs

The release experiments are carried out using six Franz cells (volume:5.68-8.88 cm³, permeation area: 0.14-0.34 cm²) which contain an isotonicglycerol solution held at 37° C. in the acceptor compartment. Theorganotypical skin construct, which is mechanically stabilised againstthe acceptor solution in a downward direction by a polycarbonate filterand is in contact with the preparation to be tested in the upwarddirection, is introduced between the donor and acceptor compartments.Samples are taken after 0; 0.5; 1; 1.5; 2; 3; 4; 5; 6 and 24 hours withreplacement of the acceptor volume of 1.0 ml removed and sent forerythropoietin analysis. A contact time of about 5 minutes, which isused for assembly of the cell, introduction of the acceptor andweighing, should be set for the period between the first contact of skinconstruct with the preparation to be tested and the first sampling (0h). Four of the release cells mentioned above are used in order to testthe formulation which, on the basis of formulation and stabilitystudies, can be regarded as the most promising formulation. In order toobtain information on whether degradation of the medicament is to befeared during the release testing owing to the elevated temperatureabove the storage conditions, two further cells are charged with aviscous solution (erythropoietin content: 150 IU/g of hydrogel) in orderto be able to determine the content from these solutions aftercompletion of the release experiments.

Example 7 EPO Analysis

Since in all cases the amounts of medicament to be determined are toosmall to carry out quantification of the non-denatured and non-degradederythropoietin content by means of optical circular dichroism,erythropoietin is determined by means of a sandwich ELISA withcompliance with the manufacturer's instructions (EPO-ELISA, OSTEOmedicalGmbH, D-Bünde). The measurement of the ELISA assay developed is carriedout using a KC4 multiplate reader (Bio-Tek, D-Bad Friedrichshall) withsoftware version 3.4 Rev 21. Since the contents of the administrationforms prepared are above the quantification range of the assay, therespective samples are diluted before measurement in order to obtain anominal content in the region of 400 mIU/ml. Samples from the releaseinvestigations can be measured without an interim dilution step.

Example 8 Skin Regeneration in Traumatised Rats Using aCarboxymethylcellulose Gel

A tangential excision trauma is made in four female rats having a bodyweight of 200-250 g. The animals are treated with a gel prepared inaccordance with the above details which comprises, as gel former,carboxymethylcellulose (3% (w/w), 42,000 mPa s) (furthermore 2.4% ofglycerol) and 150 IU of EPO/BW. The controls (placebo) comprise noactive compound. After application of the gel, the animals are providedwith a bandage or plaster. The bandage/plaster is changed every 48 h.Tissue samples are taken from the wound region after 4 and 8 days andanalysed histologically. Tests are carried out here for CD31- andnestin-positive or -negative cells by means of staining techniques knownin the prior art. The results are shown in FIGS. 2, 3 and 4. It can beseen that the re-epithelialisation is completed significantly morequickly in the EPO-containing gels (“gel E”, FIGS. 2 and 3) (on average10-50%) than in the formulations containing no active compound (“gel F”,FIGS. 2 and 3), depending on the duration of the treatment. In addition,it can be seen that, through cell-specific staining, neoformation of theepithelium generates essentially no new CD31-positive cells (FIG. 4).

Skin regeneration is investigated analogously in a 1%carboxymethylcellulose gel. The results are about 5-10% worse in thecase of the EPO-containing gel formulation than in the case of acomparable 3% gel, while no significant differences are observed in thecontrols.

Skin regeneration is investigated analogously in a 1% gel and a 3% gelbased on hydroxyethylcellulose with the same EPO concentrations.Comparable or only slightly worse results are obtained compared with thecarboxymethylcellulose gel.

Example 9

The following gel formulation for treatment of a burn wound (about 120cm²) in an adult patient was prepared by the above-mentioned process:

Constituent % by weight Hydroxyethylcellulose 250 HX Pharm 3 Glycerol(anhydrous) 2.4 Water (for injection purposes) 94.6 100 g of a gel ofthis type were applied and comprised 12,000 IU of EPO and had aviscosity of about 40,000 mPa × s.

Example 10

The following gel formulation for treatment of a cut wound (2 cm²) in anadult patient was prepared by the above-mentioned process:

Constituent % by weight Hydroxyethylcellulose 250 HX Pharm 4 Glycerol(anhydrous) 2.5 Polyacrylate 1.5 Water (for injection purposes) 92.0 2 gof a gel of this type were applied and comprised 1000 IU of EPO.Viscosity: about 68,000 mPa × s.

Example 11

The following gel formulation for treatment of a burn wound (about 300cm²) in an adult patient was prepared by the above-mentioned process:

Constituent % by weight Hydroxyethylcellulose 250 HX Pharm 2.5 Glycerol(anhydrous) 2.5 Polyacrylate 2.0 Water (for injection purposes) 93.0 450g of a gel of this type comprised 50,500 IU of EPO and were distributedon the burn wound for treatment. Viscosity: about 35,000 mPa × s.

Example 12

The following gel formulation for treatment of a burn wound (80 cm²) inan adult patient was prepared by the above-mentioned process:

Constituent % by weight Carboxymethylcellulose 250 HX Pharm 3.5 Glycerol(anhydrous) 3.5 Water (for injection purposes) 92.0 50 g of a gel ofthis type were applied and comprised 35,000 IU of EPO. Viscosity: about55,000 mPa × s.

Example 13

The following gel formulation for treatment of an open leg ulcer (150cm²) in an adult patient was prepared by the above-mentioned process:

Constituent % by weight Carboxymethylcellulose 250 HX Pharm 3.0 Glycerol(anhydrous) 2.5 Polymethacrylate 1.0 Water (for injection purposes) 93.0150 g of a gel of this type were applied and comprised 20,100 IU of EPO.Viscosity: about 49,000 mPa × s.

Example 14

The following gel formulation for treatment of a relatively large mucousmembrane/jaw wound (1 cm²) as a consequence of a tooth extraction in anadult patient was prepared by the above-mentioned process:

Constituent % by weight Carboxymethylcellulose A380 Aquasorb 3.0Glycerol (anhydrous) 2.5 Water (for injection purposes) 94.5 1 g of agel of this type was applied and comprised 1500 IU of EPO.

Example 15

The following gel formulation for treatment of a relatively large mucousmembrane/jaw wound (about 10 cm²) as a consequence of a tooth extractionin an adult patient was prepared by the above-mentioned process:

Constituent % by weight Carboxymethylcellulose A380 Aquasorb 2.0Hydroxyethylcellulose 250 HX Pharm 1.0 Glycerol (anhydrous) 2.0 Water(for injection purposes) 95.0 30 g of a gel of this type were appliedand comprised 20,500 IU of EPO. Viscosity: about 23,000 mPa × s.

Example 16 Chronic Ischaemic Wounds

Chronic ischaemic wounds are amongst the most common surgical clinicalpictures in Germany alone and require intensive interdisciplinarytreatment. In Germany, more than 3 million people suffer from chronicwounds, which can generally be divided into three types of wound: ulcuscruris, diabetic foot and decubitus. The costs for the treatment ofchronic wounds in this country are currently running at about 5 billioneuros per year. The combination of an arterial ulcer (peripheralarterial occlusive disease) and a diabetic ulcer is regarded asparticularly difficult to treat clinically and unfortunately is notrare.

A 69-year-old diabetic patient with grade IV pAVK has been sufferingfrom a grade III chronic ischaemic ulcer of the lateral malleolus formore than 12 months (FIG. 5, top picture). After three local treatmentswith recombinant EPO (in each case 3000 IU) in Varihesive E® hydrogelhaving a correspondingly high viscosity, obvious formation ofgranulation tissue occurs (middle picture). Complete healing of thewound can be observed after 15 days after a subsequent split-skin graft(bottom picture).

Example 17 Dermal Wounds

Everyone will suffer from a certain number of mechanical or thermaldermal injuries which require healing in the course of his or her life.The market for local wound therapeutic agents in the form of liquids,ointments or bandage materials is correspondingly large. In theevidence-based analysis of affected patients, the fact that each skinwound has a different configuration is problematical. However, insurgery there is a highly standardised skin wound which is produced bythe need to remove a part of the skin of defined thickness (usually 0.2to 0.3 mm) using a special instrument (dermatome) in order then to graftit to another site in the body. Split-skin removal sites of this type(0.3 mm) usually heal without consequences within 10-14 days.

A 25-year-old patient had a thermal injury whose treatment madesplit-skin removal necessary (FIG. 6, left-hand picture). The removalsites were treated with 1×3000 IU of EPO in hydrogel in accordance withone of the compositions indicated above (3% of carboxymethylcellulose).Complete healing was observed after only 7 days (FIG. 6, right-handpicture).

Similar results were achieved with four further patients who hadsuffered burns.

The therapy trial of split-skin graft removal sites with locally appliederythropoietin was carried out as follows on the said five patients: thesplit-skin grafts were each taken from the thigh with a thickness of 0.3mm. The erythropoietin was mixed intraoperatively with Varihesive®hydrogel, which functioned as carrier substance, and applied directly tothe wound. The wound area was subsequently covered with a perforatedpolyurethane film and a secondary bandage. The secondary bandage wasonly removed in the case of further local erythropoietin applications.The latter were carried out in a sterile manner, at a number of points,through the polyurethane film. The polyurethane film was only removed,atraumatically, seven days after the operation in order to assessre-epithelialisation.

1-40. (canceled)
 41. A gelatinous or viscous formulation comprisingerythropoietin (EPO) or one of its derivatives or analogues having thesame biological action, and at least one gel-forming, swellablepolysaccharide in a concentration of 0.4 to 4% by weight selected fromone or more members of the group consisting of: hydroxyethylcellulosehydroxymethylcellulose carboxyethylcellulose carboxymethylcellulose,obtained by mixing EPO in lyophilised, dissolved or suspended form withthe pre-swollen polysaccharide having a viscosity of less than 5000mPa×s and initiation of complete swelling, wherein the fully swollenpolysaccharide has a viscosity of 20,000-60,000 mPa×s, and EPO ispresent in a concentration of 100-500 IU/g of the gelatinousformulation.
 42. A formulation according to claim 41, wherein saidmixing of EPO is achieved by diffusing EPO into the gel for at least 24h and rotating the gel twice through 180° within said period.
 43. Aformulation according to claim 41, characterised in that theconcentration of the polysaccharide(s) is 2-3% by weight.
 44. Aformulation according to claim 41, characterised in that the gel-formingpolysaccharide is carboxymethylcellulose containing 150 IU of EPO/g ofgelatinous formulation, and 1 g of gel/cm² of wound area is employed.45. A formulation according to one of claim 41, characterised in that ithas been introduced into or onto a solid carrier matrix which causes EPOto be released uniformly into the wound region of the traumatised skin.46. A formulation according to claim 45, characterised in that the solidcarrier matrix is a three-dimensionally structured plaster.
 47. A methodof using the formulation of claim 41 for the preparation of a medicamentfor the topical and local treatment of diseased or traumatized skin ofburn wounds, scalds, chronically ischaemic wounds and in the dentalarea.
 48. The method of claim 47, characterised in that 50-1.500 IUEPO/cm² wound area is applied.
 49. The method of claim 48, characterisedin that 75-450 IU EPO/cm² wound area is applied.
 50. A process for themanufacture of a stabile erythropoietin (EPO) containing gelatinousformulation comprising diffusing EPO into a pre-swollen gel for at least24 h, the gel comprising a polysaccharide selected from one or moremembers of the group consisting of hydroxyethylcellulose,hydroxymethylcellulose, carboxyethylcellulose, carboxymethylcellulose,hydroxylpropylcellulose, and carboxypropylcellulose in a concentrationof 0.4-4 per weight.
 51. The process according to claim 50,characterised in that the gel is rotated through 180° during the periodof diffusion.
 52. The process according to claim 51, characterised inthat the gel is rotated twice.
 53. The process according to claim 50,characterised in that the concentration of the polysaccharide is 1.5 to3.5% by weight.